Fetal alcohol spectrum disorder (FASD) is caused by prenatal alcohol exposure, resulting in craniofacial dysmorphology, cognitive impairment, sensory disabilities, motor disabilities and organ deformities. FASD occurs in 3-10% of children born in US populations. The long-term goal of this project is to understand the molecular mechanisms and to identify therapeutic targets for FASD. The overall hypothesis is that ethanol exposure produces detrimental epigenetic and gene expression effects that disrupt critical early embryogenesis events, which are rescued by folic acid (FA) treatment. Early development (gastrulation) is a highly sensitive period for ethanol-induced defects, and in humans, gastrulation occurs at implantation, the first possible time to be exposed to maternal circulation and thus, maternal blood alcohol. Developmental gene expression and epigenetic events are highly conserved in vertebrates. Our data point to ethanol and FA- sensitive gastrulation events occurring in early zebrafish embryos. FA is part of one-carbon metabolism pathways that facilitate methyl group addition to DNA and histones. Understanding FA protective effects will help us better target birth defect prevention strategies and potentially identify new therapeutic strategies. Our preliminary gene expression studies showed that Sox2 and Elf3 transcription factors are sensitive to ethanol exposure in the early embryo and are parts of an extensive transcriptional network. Specific aim 1: Determine genome-wide changes in DNA-methylation, chromatin structure and gene expression in response to early ethanol and FA exposure. Proposed experiments will analyze genome-wide ethanol and FA effects on DNA- methylation (MeDIP-seq), chromatin structure (ATAC-seq) and gene expression data (RNA-seq) collected in parallel. This dataset will determine whether ethanol affects epigenetic events, gene expression and whether FA can prevent these epigenetic defects. Interrogating histone modifications at specific gene promoters by targeted chromatin immunoprecipitation and quantitative PCR (ChIP-PCR) will analyze epigenetic mechanisms occurring during large chromatin structural changes (ATAC-seq). Specific aim 2: Analyze potential gene interactions between sox2 and elf3 during ethanol-induced embryogenesis defect genesis. Proposed experiments will evaluate individual roles of sox2 and elf3 and potential interactions between sox2 and elf3 during early development and determine whether these interactions have functional significance in our FASD model. This overall project has the expertise to get robust answers about ethanol's role in epigenetic events, gene expression programs and functional consequences. The mechanism of action for ethanol during early development is a knowledge gap, and this study will examine epigenetic effects in parallel with gene expression effects. Future therapeutic approaches will rely on our knowledge of the birth defect syndrome's genesis.